Cholinergic deficits and galaninergic hyperinnervation of the nucleus basalis of Meynert in Alzheimer's disease and Lewy body disorders.

AIMS: Galanin is a highly inducible neuroprotective neuropeptide and in Alzheimer's disease (AD), a network of galaninergic fibres has been reported to hypertrophy and hyperinnervate the surviving cholinergic neurons in the basal forebrain. We aimed to determine (i) the extent of galanin hyperinnervation in patients with AD and Lewy body disease and (ii) whether galanin expression relates to the neuropathological burden and cholinergic losses. METHODS: Galanin immunohistochemistry was carried out in the anterior nucleus basalis of Meynert of 27 Parkinson's disease (PD) cases without cognitive impairment (mild cognitive impairment [MCI]), 15 with PD with MCI, 42 with Parkinson's disease dementia (PDD), 12 with Dementia with Lewy bodies (DLB), 19 with AD, 12 mixed AD/DLB and 16 controls. Galaninergic innervation of cholinergic neurons was scored semiquantitatively. For a subgroup of cases (n = 60), cholinergic losses were determined from maximum densities of choline acetyltransferase positive (ChAT+ve) neurons and their projection fibres. Quantitative data for α-synuclein, amyloid beta and tau pathology were obtained from tissue microarrays covering cortical/subcortical regions. RESULTS: Significant losses of cholinergic neurons and their projection fibres were observed across all diseases. Galaninergic hyperinnervation was infrequent and particularly uncommon in established AD and DLB. We found that hyperinnervation frequencies are significantly higher in the transition between PD without MCI to PDD and that higher burdens of co-existent AD pathology impair this galaninergic response. CONCLUSIONS: Our results suggest that galanin upregulation represents an intrinsic response early in Lewy body diseases but which fails with increasing burdens of AD related pathology.

Review: Revisiting the human cholinergic nucleus of the diagonal band of Broca.

Although the nucleus of the vertical limb of the diagonal band of Broca (nvlDBB) is the second largest cholinergic nucleus in the basal forebrain, after the nucleus basalis of Meynert, it has not generally been a focus for studies of neurodegenerative disorders. However, the nvlDBB has an important projection to the hippocampus and discrete lesions of the rostral basal forebrain have been shown to disrupt retrieval memory function, a major deficit seen in patients with Lewy body disorders. One reason for its neglect is that the anatomical boundaries of the nvlDBB are ill defined and this area of the brain is not part of routine diagnostic sampling protocols. We have reviewed the history and anatomy of the nvlDBB and now propose guidelines for distinguishing nvlDBB from other neighbouring cholinergic cell groups for standardizing future clinicopathological work. Thorough review of the literature regarding neurodegenerative conditions reveals inconsistent results in terms of cholinergic neuronal loss within the nvlDBB. This is likely to be due to the use of variable neuronal inclusion criteria and omission of cholinergic immunohistochemical markers. Extrapolating from those studies showing a significant nvlDBB neuronal loss in Lewy body dementia, we propose an anatomical and functional connection between the cholinergic component of the nvlDBB (Ch2) and the CA2 subfield in the hippocampus which may be especially vulnerable in Lewy body disorders.

A novel method to visualise the three-dimensional organisation of the human cerebral cortical vasculature.

Current tissue-clearing protocols for imaging in three dimensions (3D) are typically applied to optimally fixed, small-volume rodent brain tissue - which is not representative of the tissue found in diagnostic neuropathology laboratories. We present a method to visualise the cerebral cortical vasculature in 3D in human post-mortem brain tissue which had been preserved in formalin for many years. Tissue blocks of cerebral cortex from two control cases, two Alzheimer's brains and two cases from Alzheimer's patients immunised against Aß42 were stained with fluorescent Lycopersicon esculentum agglutinin (Tomato lectin), dehydrated and cleared using an adapted three-dimensional imaging of solvent cleared organs (3DISCO) protocol to visualise the vascular endothelium. Tissue was imaged using light sheet and confocal microscopy and reconstructed in 3D using amira software. The method permits visualisation of the arrangement of the parallel penetrating cortical vasculature in the human brain. The presence of four vascular features including anastomosis, U-shaped vessels, spiralling and loops were revealed. In summary, we present a low cost and simple method to visualise the human cerebral vasculature in 3D compatible with prolonged fixation times (years), allowing study of vascular involvement in a range of normative and pathological states.

Bringing CLARITY to the human brain: visualization of Lewy pathology in three dimensions.

AIMS: CLARITY is a novel technique which enables three-dimensional visualization of immunostained tissue for the study of circuitry and spatial interactions between cells and molecules in the brain. In this study, we aimed to compare methodological differences in the application of CLARITY between rodent and large human post mortem brain samples. In addition, we aimed to investigate if this technique could be used to visualize Lewy pathology in a post mortem Parkinson's brain. METHODS: Rodent and human brain samples were clarified and immunostained using the passive version of the CLARITY technique. Samples were then immersed in different refractive index matching media before mounting and visualizing under a confocal microscope. RESULTS: We found that tissue clearing speed using passive CLARITY differs according to species (human vs. rodents), brain region and degree of fixation (fresh vs. formalin-fixed tissues). Furthermore, there were advantages to using specific refractive index matching media. We have applied this technique and have successfully visualized Lewy body inclusions in three dimensions within the nucleus basalis of Meynert, and the spatial relationship between monoaminergic fibres and Lewy pathologies among nigrostriatal fibres in the midbrain without the need for physical serial sectioning of brain tissue. CONCLUSIONS: The effective use of CLARITY on large samples of human tissue opens up many potential avenues for detailed pathological and morphological studies.